Shotgun Sequencing of Bacteria from AHPNS A New Shrimp ...

twoeggfinnishΒιοτεχνολογία

14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

585 εμφανίσεις

Shotgun Sequencing of Bacteria from AHPNS

A New Shrimp Disease Threat for Thailand

SUMMARY

The

most

threatening

new

problem

in

Asian

shrimp

aquaculture

at

this

time

is

serious

mortality

from

unknown

causes

that

began

in

China

in

2009
,

then

Vietnam

since

2010

and

more

recently

in

2011

to

the

eastern

coast

of

Malaysia

and

the

eastern

coast

of

the

gulf

of

Thailand
.

So

far

no

potential

causative

pathogen

has

been

found

and

possible

etiologies

include

toxins

(biotic

or

abiotic),

bacteria

and

viruses
.

A

case

definition

for

the

syndrome

affecting

shrimp

in

the

outbreak

ponds

has

been

described

by

D
.
V
.

Lightner

from

the

University

of

Arizona

(unpublished)

and

this

is

consistent

with

the

specimens

that

have

been

received

and

analyzed

by

Centex

Shrimp

in

Thailand

(T
.
W
.

Flegel
,

unpublished)
.

Dr
.

Lightner

has

referred

to

this

syndrome

as

acute

hepatopancreatic

necrosis

syndrome

(AHPNS)

that

can

be

described

as

acute

progressive

degeneration

of

the

hepatopancreas

(HP)

with

the

following

histological

features
:

lack

of

mitotic

activity

in

generative

E

cells

of

the

HP
;

central

dysfuncton

of

hepatopancreatic

B,

F

and

R

cells
;

prominent

karyomegaly

and

massive

sloughing

of

central

HP

tubule

epithelial

cells
;

terminal

stages

including

massive

untertubular

hemocytic

aggregation

followed

by

secondary

bacterial

infections
.

To

test

for

the

possible

involvement

of

bacteria

in

causing

AHPNS,

we

carried

out

shotgun

sequencing

of

bacterial

ssrDNA

fragments

amplified

from

HP

of

shrimp

from

AHPNS

ponds
.

From

analysis

of

approximately

100
,
000

fragments

from

6

disease

ponds

and

one

control

pond,

a

total

of

8327

unique

sequences

(taxonomic

units

or

TU
)

of

approximately

400

bp

were

obtained
.

Eliminating

singleton

TU

left

1205

TU

with

reads

from

2

or

more

ponds
.

A

comparison

of

the

read

frequency

for

these

yielded

5

TU

with

the

highest

difference

between

test

ponds

and

the

control

pond
.

These

sequences

will

be

used

to

design

specific

probes

for

in

situ

hybridization

assays

with

HP

tissues

from

AHPNS

shrimp
.


INTRODUCTION

Since

the

serious

mortality

from

unknown

causes

that

began

in

China

in

2009
,

there

have

been

rumors

that

infectious

myonecrosis

(IMN)

caused

by

the

virus

IMNV

may

have

been

the

cause
.

IMNV

was

first

described

from

Indonesia

(
Senapin

et

al
.

2007
.

Aquaculture

266
:
32
-
38
)

as

a

probable

example

for

disease

translocation

with

aquaculture

stocks

from

Brazil
.

However,

continual

testing

at

Centex

Shrimp

has

revealed

that

these

have

been

false

rumors

(
Senapin

et

al
.

2011
.

BMC

J

Negative

Results

in

Biomedicine
.

10
:
10
)
.

Most

of

these

rumors

were

probably

caused

by

muscle

cramp

syndrome

that

can

commonly

cause

whitened

muscles

in

stressed

Penaeus

(
Litopenaeus
)

vannamei
.

Addi t onal

r umor s

may

have

resulted

from

light,

false
-
positive

RT
-
PCR

results

using

the

IQ
2000

test

kit

with

DNA

extracts

from

shrimp

pleopods

rather

than

excised,

infected

muscle

tissue

or

hemolymph

(i
.
e
.
,

internal

material)
.

Since

samples

from

the

outbreaks

in

Vetnam

and

China

have

tested

negative

for

IMNV

by

RT
-
PCR,

histopathology

and

immunohistoghemistry
,

IMNV

can

be

ruled

out

as

the

cause

of

the

problems

in

Vietnam

and

China
.



In

addition,

other

pathogens

newly

discovered

in

P
.

vannamei

from

Thailand

and

Vietnam

[including

Macrobrachium

rosenbergii

nodavirus

(
MrNV
)

and

the

microsporidian

Enterocytozoon

hepatopenaei
]

have

also

proven

not

to

be

the

cause

of

the

massive

disease

outbreaks

based

on

low

prevalence

in

tested

specimens

from

the

outbreak

ponds

as

revealed

by

histological

analysis,

PCR

testing

and

immunohistochemistry
.

In

addition,

tests

for

nectotizing

hepatopancreatic

necrosis

(NHP)

have

proven

negative

by

PCR

testing,

histological

analysis

and

transmission

electron

microscopy

(TEM)
.



W
e

are

cooperating

with

Dr
.

Niti

Chuchird

and

Dr
.

Chalor

Limsuwan

at

Kasetsart

Universty

to

investigate

the

possibility

of

novel

bacteria

that

might

be

present

in

shrimp

from

AHPNS

ponds

using


shotgun”

samples

originating

from

normal

ponds

and

AHPNS

ponds
.

Since

this

is

a

new

technological

approach,

we

have

already

begun

with

the

simplest

method

of

screening

for

bacterial

pathogens
.

Briefly,

the

process

involves

use

of

universal

bacterial

primers

to

amplify

a

500

bp

small

subunit

ribosomal

RNA

(
ssu

rRNA
)

gene

fragment

from

all

bacteria

present

in

shrimp

HP

tissue

samples
.

These

are

then

sequenced

in

mass

followed

by

computer

analysis

to

collate

the

sequences
.

The

objective

is

to

be

able

to

compare

the

sequences

from

normal

and

disease

ponds

to

search

for

any

unusual

bacteria

characteristic

of

the

disease

ponds

only
.



RESULTS

Sequencing

and

bioinformatics

Using

sets

of

shrimp

pooled

from

each

of

7

ponds

(
3

diseased

and

one

control

pond

from

Vietnam,

and

5

diseased

ponds

from

Thailand),

DNA

was

extracted

and

used

as

the

template

for

PCR

amplification

of

an

rRNA

gene

fragment

of

an

expected

size

of

approximately

500

bp
.

The

amplified

bands

from

each

pond

were

cut

from

the

agarose

gel,

eluted

from

the

gel

and

subjected

to

DNA

barcode

addition

for

each

pond

sample
.

Next,

the

pooled

samples

were

subjected

to

Roche

454

sequencing
.

A

total

number

of

41

million

bases

were

read

with

an

average

read

of

450

bp

yielding

approximately

100
,
000

sequences
.

At

97
%

identity

for

sequences

of

approximately

400
-
500

bp

with

primers

at

both

ends,

a

total

of

8327

unique

sequences

(taxonomic

units

or

TU)

were

obtained
.

Of

these,

7123

TU

were

singletons

(i
.
e
.
,

arising

from

only

one

pond

as

single

or

as

multiple

reads)
.



Eliminating

the

singleton

TU

left

1205

TU

with

reads

from

2

or

more

ponds
.

A

comparison

of

the

read

frequency

for

each

of

the

1205

TU

for

each

of

the

ponds

yielded

the

following

5

TU

with

the

highest

difference

between

test

ponds

and

the

control

pond
.

The

genus

names

are

numbered

and

shown

in

yellow

with

example

species

names

for

Blast

hits

given

in

brackets
.

The

latter

may

or

may

not

be

the

actual

species

in

the

target

samples
.



Order

Burkholderiales
:



Family

Burkholderiaceae



Genus

Ralstonia

(
1
)


Family

Comamonadaceae

(all

found

were

former

Pseudomonas

species)



Genus

Delftia

(
acidovorans

&

tsuruhatensis
)

(
2
)



Genus

Pelomonas

(
aquatica
)

(
3
)



Order

Sphingomonadales

Order

Actinomycetales


Family

Microbacteriaceae



Genus

Leifsonia

(
4
)


Family

Nocardiaceae



Genus

Rhodococcus

(
globerulus
,

erythropolis
,

boritolerans
)

(
5
)


karyomegaly

AHPNS
histopathology


Sloughing
of HP cells



Lack
of E
-
cell mitosis

Lack
of B, F &
R cells



Enlarged
HP nuclei

Hemocytic

infiltration

2
o

bacterial infection


Semi
-
thin HP tissue sections

Thai

samples

of

P
.

vannamei


collected

from

Chantaburi

and

Rayong

provinces

in

late

2011

and

early

2012

showed

histological

signs

of

AHPNS

and

HP

tissues

were

embedded

in

epoxy

resin

to

prepare

for

transmission

electron

microscopy
.

In

prelmnary

semi
-
thin

sections

stained

with

toluidine

blue,

abnormal

blebbing

of

the

HP

tubule

margins

was

seen
.

Since

it

occurred

in

many

tubules

in

the

absence

of

visible

bacteria,

supportng

Dr
.

Lightner’s

earlier

proposal

for

a

possible

toxic

origin
.

This

phenomenon

was

also

seen

in

similar

HP

tissue

sections

of

AHPNS

shrimp

specimens

from

Vietnam

and

Malaysia,

indicating

that

the

phenomenon

could

serve

as

an

additional

characteristic

of

the

AHPNS

case

definition
.



















CONCLUSIONS

The

bacterial

sequences

obtained

from

AHPNS

shrimp

by

shotgun

sequencing

were

not

related

to

sequences

normally

reported

from

diseased

shrimp,

particularly

with

respect

to

Vbrio

speci es
.

Of

the

genera

showing

significant

homology,

Delftia

was

the

most

prevalent

in

our

samples,

but

aside

from

one

report

for

a

new

species

(
Delftia

litopenaei
)

described

from

normal

shrimp

in

Taiwan

(Chen

et

al
.

2011
.

IJSEM,

in

press

doi
:
10
.
1099
/ijs
.
0
.
037507
-
0
),

no

species

of

Delftia

has

previously

been

reported

from

shrimp
.

Similarly,

none

of

the

other

4

genera

from

the

homology

search

have

previously

been

reported

from

shrimp

and

all

5

are

most

frequently

reported

from

water

or

wastewater

samples
.

Their

possible

role

in

AHPNS

will

be

assessed

by

preparation

of

labeled

probes

for

in

situ

hybridization

tests

using

AHPNS

shrimp

samples
.


Acknowledgements

This

work

was

supported

by

Mahidol

Universty
,

Charoen

Pokphand

Co
.

Ltd
.
,

the

Higher

Education

Research

Promotion

and

National

University

Development,

Office

of

the

Thailand

Higher

Education

Commission,

the

Surathani

Shrimp

Farmer’s

Club,

the

Thai

Frozen

Foods

Association

and

the

National

Research

Council

of

Thailand
.



Anuparp

Prachumwat
1
,
2
,

Siripong

Thitamadee
1
,
3
,

Siriporn

Sriurairatana
1
,

Niti

Chuchird
4
,

Chalor

Limsuwan
4
,

Wassan

Jantratit
5
,

Sage

Chaiyapechara
6
,

Timothy

W
.

Flegel
1
,
6



1
Centex

Shrimp,

Faculty

of

Science,

Mahidol

University,

Rama

6

Road,

Bangkok

10400
,

Thailand

2
Shrimp
-
Virus

Interaction

Laboratory,

National

Center

for

Genetic

Engineering

and

Biotechnology

(BIOTEC
),

National

Science

and

Technology

Development

Agency,

Klong

1
,

Klong

Luang
,

Pathumthani

12120
,

Thailand

3
Department

of

Biotechnology,

Faculty

of

Science,

Mahidol

University,

Rama

6

Road,

Bangkok

10400
,

Thailand

4
Department

of

Fishery

Biology,

Faculty

of

Fisheries,

Kasetsart

University,

Pahonyothin

Rd
.
,

Bangkok

10900
,

Thailand

5
Faculty

of

Medicine,

Ramathibodi

Hospital,

Mahidol

University,

Rama

6
,

Road,

Bangkok

10400
,

Thailand

6
National

Center

for

Genetic

Engineering

and

Biotechnology

(BIOTEC
),

National

Science

and

Technology

Development

Agency,

Klong

1
,

Klong

Luang
,

Pathumthani

12120
,

Thailand